1. Field of the Invention
The present invention relates to electrochemical control of bacterial cells and, more particularly, the effect of weak electric currents on bacterial cells.
2. Description of the Related Art
Previous studies of persister cells have led to important discoveries that are shifting the paradigm of research in microbiology and antimicrobial therapy. It is now well recognized that subpopulations of bacterial cells in a culture can enter a dormant (non-growing) state that are extremely tolerant to a variety of unrelated stresses such as antibiotics and heat. Such heterogeneity has been reported to exist in even well mixed shake flask cultures at exponential phase. This phenotypic variation can lead to three subpopulations in a given culture including the normal cells, type I persister cells from the stationary inoculums and type II persister cells that are generated during growth. Persister cells are not mutants with drug resistant genes, but rather phenotypic variants of the wild-type strain. Persister cells neither die nor grow in the presence of an antibiotic, and when reinoculated, they grow into a normal culture with a similar percentage of cells as persisters, leading to high antibiotic tolerance.
Although persister cells normally only make up a small portion of the population, they play a critical role in antibiotic tolerance. Most antibiotics inhibit bacteria by targeting growth related cellular activities, e.g., protein, DNA, and cell wall syntheses. They can eliminate the majority of bacterial population by killing the normal cells. For persister cells, however, antibiotics can only repress but not eliminate this subpopulation because persister cells are non-growing dormant cells. Thus, the seeming disadvantage of being dormant in normal environment becomes an advantage for persister cells when being challenged by antibiotics. When the treatment is stopped, some persister cells revert back to normal cells and reestablish the population. Such tolerance leads to reoccurrence of infections and facilitate the development and spread of multidrug resistance through true mutations.
Recent research has demonstrated that persister cell formation increases significantly in stationary-phase cultures and the surface-attached highly hydrated structures known as biofilms. Formed in a dynamic process, mature biofilms typically have mushroom-like structures with cells embedded in a polysaccharide matrix secreted by the bound bacterial cells. Biofilm cells are up to 1000 times more tolerant to antibiotics and disinfectants compared to their planktonic counterparts. Thus, deleterious biofilms cause serious problems such as chronic infections in humans as well as persistent corrosion and equipment failure in industry. Although not completely understood at the molecular level, the biofilm-associated tolerance is due to several factors acting in concert. Bacterial cells in biofilm produce a polysaccharide matrix, which creates a physical barrier that retards or blocks the toxic compounds from reaching the cells. However, protection by the polysaccharide matrix can only partially explain the tolerance because at least some antibiotics can readily penetrate the matrix yet still can not eliminate biofilm cells. Biofilm mode of growth is also associated with changes in bacterial membrane structure and reduction in cell growth rate. The changes in membrane structure could reduce the permeability to toxic compounds, while the reduction in growth rate can lead to higher tolerance to growth-dependent killing by antibiotics. Increasing evidence suggests that the slow growth, especially that associated with persister cells, is the most challenging mechanism for treating chronic infections.
The rapid development and spread of multidrug resistant infections present an increasing challenge to public health and disease therapy. As an intrinsic mechanism of drug resistance, biofilm formation renders bacteria up to 1000 times less susceptible to antibiotics than their planktonic (free-swimming) counterparts of the same genotype. Such intrinsic resistance also facilitates the development of resistance through acquired mechanisms that are based on genetic mutations or drug resistance genes. Consistently, excessive antibiotic treatment of biofilm infections at sublethal concentrations has been shown to generate antibiotic-tolerant strains. It is estimated that biofilms are responsible for at least 65% of human bacterial infections. For example, it is estimated that in the United States 25% of urinary catheters become infected with a biofilm within one week of a hospital stay, with a cumulative 5% chance each subsequent day. Biofilms are also detected on implanted devices and are a major cause of explanation. Orthopedic implants showed a 4.3% infection rate, or approximately 112,000 infections per year in the U.S. This rate increases to 7.4% for cardiovascular implants, and anywhere from 5%-11% for dental implants.
In the biofilm state, bacteria undergo significant changes in gene expression leading to phenotypic changes that serve to enhance their ability to survive challenging environments. Although not completely understood, the tolerance to antibiotic treatments is thought to arise from a combination of limited antibiotic diffusion through the extracellular polymeric substances (EPS), decreased growth rate of biofilm cells, and increased expression of antibiotic resistance genes in biofilm cells (10). Treatments that are capable of removing biofilms from a surface are by necessity harsh and often unsuitable for use due to medical or environmental concerns. It is evident that alternative methods of treating bacterial infections, and most notably biofilms, are required.
Electric currents/voltages are known to affect cells. However, most of the studies have been focused on high voltages and current levels such as eletctroporation, electrophoresis, iontophoresis, and electrofusion except for a few studies about biofilm control using weak electric currents. In 1994, Costerton and colleagues reported an interesting synergistic effect between low level direct currents (DCs) and tobramycin in killing Pseudomonas aeruginosa biofilm cells grown in a continuous-flow chamber. This synergistic phenomenon was termed the “bioelectric effect.” In addition to P. aeruginosa, bioelectric effects have also been reported for Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, P. fluorescens, as well as mixed species biofilms. Although the impact of electric currents on bacterial susceptibility to antibiotics and biocides is well accepted, there is little understanding about the mechanism of bioelectric effect.
An electric current at an electrode surface can trigger ion flux in the solution as well as electrochemical reactions of the electrode materials and redox species with electrolyte and generate many different chemical species, e.g. metal ions, H+ and OH−. Although pH change has been shown to cause contraction of the biofilm formed on the cathodic electrode, change of medium pH to which prevails during electrolysis did not enhance the activity of antibiotics. Consistent with this observation, buffering the pH of the medium during electrolysis fails to eliminate bioelectric effect. Another finding suggesting the existence of other factors is that the bioelectric effect has been observed for biofilms formed in the middle of an electric field, but not in contact with either the working electrode or counter electrode. Since the electrochemically-generated ions accumulate around the electrodes, the biofilms in the middle of an electric field are not experiencing significant changes in pH or other products of electrochemical reactions. This is also evidenced by the report that radio frequency alternating electric current can enhance antibiotic efficacy. Since no electrochemically generated molecules or ions will likely accumulate with alternating currents, other factors may play a critical role.
The bioelectric effect was also observed when the growth medium only contained glucose and two phosphate compounds. This observation eliminates the electrochemical reaction of salts as an indispensable factor of bioelectric effect. Previous studies have also ruled out the impact of temperature change during electrolysis (less than 0.2° C.). Although these studies provided useful information about bioelectric effect, its mechanism is still unknown. The exact factors causing bioelectric effect and their roles in this phenomenon remain elusive. Compared to biofilms, even less is known about the effects of weak electric currents on planktonic cells.
It is important to note that many aspects of cellular functions are electrochemical in nature. That is, the redox state of cells is related to membrane status, oxidative status, energy generation and utilization and other factors. Therefore, it is possible that redox state of cells may be affected by electrochemical currents (henceforth ECs). To better understand the effects of ECs on planktonic and biofilm cells, we conducted a systematic study of the effects of weak ECs on the planktonic and biofilm cells of the model Gram-positive bacterium Bacillus subtilis. Gram-positive bacteria are responsible for 50% of infections in the United States, and 60% of nosocomial infections. With the emergence and wide spread of multidrug resistant bacteria, effective methods to eliminate both planktonic bacteria and those embedded in surface-attached biofilms are badly needed.